Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D233/20—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with substituted hydrocarbon radicals, directly attached to ring carbon atoms
  • C07D233/22—Radicals substituted by oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D233/20—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with substituted hydrocarbon radicals, directly attached to ring carbon atoms
  • C07D233/26—Radicals substituted by carbon atoms having three bonds to hetero atoms

Definitions

• This invention is related to at least one compound selected from a compound of formula I
• p53 is a tumor suppresser protein that plays a central role in protection against development of cancer. It guards cellular integrity and prevents the propagation of permanently damaged clones of cells by the induction of growth arrest or apoptosis.
• p53 is a transcription factor that can activate a panel of genes implicated in the regulation of cell cycle and apoptosis.
• p53 is a potent cell cycle inhibitor which is tightly regulated by MDM2 at the cellular level. MDM2 and p53 form a feedback control loop. MDM2 can bind p53 and inhibit its ability to transactivate p53-regulated genes. In addition, MDM2 mediates the ubiquitin- dependent degradation of p53.
• MDM2 is also a cofactor for E2F, which plays a central role in cell cycle regulation.
• E2F The ratio of MDM2 to p53 (E2F) is dysregulated in many cancers. Frequently occurring molecular defects in the pl6INK4/pl9ARF locus, for instance, have been shown to affect MDM2 protein degradation. Inhibition of MDM2-p53 interaction in tumor cells with wild-type p53 should lead to accumulation of p53, cell cycle arrest and/or apoptosis.
• MDM2 antagonists therefore, can offer a novel approach to cancer therapy as single agents or in combination with a broad spectrum of other antitumor therapies.
• the feasibility of this strategy has been shown by the use of different macromolecular tools for inhibition of MDM2-p53 interaction (e.g. antibodies, antisense oligonucleotides, peptides).
• MDM2 also binds E2F through a conserved binding region as p53 and activates E2F-dependent transcription of cyclin A, suggesting that MDM2 antagonists might have effects in p53 mutant cells.
• EP 363 061 to Matsumoto reports imidazoline derivatives useful as immunomodulators. The compounds were indicated to have low toxicity. Treatment and/or prevention of rheumatoid arthritis, multiple sclerosis, systemic lupus, erythemathodes, and rheumatic fever were implicated.
• WO 00/78725 to Choueiry et al. report a method for making substituted amidine compounds, and indicate that imidazoline-type compounds may be useful in the treatment of diabetes or related diseases involving impaired glucose disposal.
• the present invention provides chiral cis-imidazolines which are small molecule inhibitors of the MDM2-p53 interaction.
• compounds of the present invention are shown to inhibit the interaction of MDM2 protein with a p53-like peptide with a potency that is approximately 100 fold greater than a p53 -derived peptide.
• these compounds demonstrate mechanistic activity. Incubation of cancer cells with wild-type p53 leads to accumulation of p53 protein, induction of p53-regulated p21 gene, and cell cycle arrest in G 1 and G2 phase, resulting in potent antiproliferative activity against wild-type p53 cells in vitro. In contrast, these activities were not observed in cancer cells with mutant p53 at comparable compound concentrations. Therefore, the activity of MDM2 antagonists is likely linked to its mechanism of action. These compounds can be potent and selective anticancer agents.
• the present invention provides at least one compound of formula I
• Xi is selected from the group consisting of lower alkoxy, and lower alkoxy substituted by trifluoromethyl or fluorine;
• X 2 is selected from the group consisting of hydrogen, halogen, lower alkyl, and -C(X 4 X 5 )-X 6 ;
• X 3 is selected from the group consisting of hydrogen, lower alkoxy, halogen, and -C(X X 5 )-X 6 ; with the proviso that when X 2 is hydrogen, halogen or lower alkyl, X 3 is -C(X 4 X 5 )-X 6 ;
• X 4 and X 5 are lower alkyl and can be connected together to form a cycloalkyl
• X 6 is selected from the group consisting of lower alkyl, cyano, -CH 2 -OH, -CH 2 -O-lower alkyl, -CH 2 -O-lower alkyl substituted by lower alkoxy, -C(O)X 7 , and -CH 2 -NX 8 X 9 ;
• X 7 is selected from the group consisting of hydroxy, lower alkoxy, morpholino, and -NX 8 X ;
• X 8 and X 9 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkyl substituted by lower alkoxy or cyano, and lower alkoxy;
• Yi and Y 2 are independently selected from the group consisting of halogen, cyano, and acetylene;
• R 2 is selected from the group consisting of hydroxy, lower alkoxy, trifluoromethyl, -cyano, - NH-SO 2 -lower alkyl, -NH-C(O)-lower alkyl, -C(O)-lower alkyl, -C(O)R 4 , -C(O)-NX 8 X 9 , -SO 2 - lower alkyl, -SO 2 -NX 8 X 9 ,
• R 3 is selected from the group consisting of five membered heterocycle, lower alkyl, lower alkoxy, and lower alkyl substituted by lower alkoxy;
• R 4 is selected from the group consisting of hydroxy, lower alkoxy, morpholino, and -NX 8 X 9 ;
• Preferred compounds are compounds of formula I wherein Yi and Y are each independently selected from -CI and -Br.
• Also preferred compounds are compounds in which the two hydrogen atoms of the imidazoline ring are in a cis configuration to each other.
• the compounds may be in a racemic form and may be optically active.
• the absolute stereochemistry at the imidazoline ring of formula I is S at the 4-position and R at the 5-position.
• Such compounds are for example: [(4S,5R)-2-(4-tert-Butyl-2-ethoxy-phenyl)-4,5-bis-(4-fluoro-phenyl)-4,5-dihydro- imidazol- 1 -yl]-[4-(2-methanesulfonyl-ethyl)-piperazin- 1 -yl]-methanone hydrochloride; 4-[(4S,5R)-2-(4-tert-Butyl-2-ethoxy-phenyl)-4,5-bis-(4-fiuoro-phenyl)-4,5-dihydro- imidazole- 1 -carbonyl] -piperazin-2-one hydrochloride; 2- ⁇ 4-[(4S,5R)-2-(4-tert-Butyl-2-ethoxy-phenyl)-4,5-bis-(4-fluoro-phenyl)-4,5-dihydro- imidazole-
• Ri is selected from
• Such compounds are for example: 2- ⁇ 4-[(4S,5R)-2-(4-tert-butyl-2-ethoxy-phenyl)-4,5-bis-(4-chloro-phenyl)-4,5-dihydro- imidazole-l-carbonyl]-piperazin-l-yl ⁇ -l-mo ⁇ holin-4-yl-ethanone hydrochloride; and [(4S,5R)-2-(4-tert-butyl-2-ethoxy-phenyl)-4,5-bis-(4-chloro-phenyl)-4,5-dihydro- imidazol-l-yl]-[4-(2-methanesulfonyl-ethyl)-piperazin-l-yl]-methanone hydrochloride.
• Effective amount means an amount that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
• Halogen means fluorine, chlorine, bromine or iodine.
• Hetero atom means an atom selected from N, O and S.
• IC 50 refers to the concentration of a particular compound required to inhibit 50% of a specific measured activity. IC 50 can be measured, inter alia, as is described subsequently.
• Alkyl denotes a straight-chained or branched saturated aliphatic hydrocarbon.
• Cycloalkyl means a non-aromatic, partially or completely saturated monovalent cyclic hydrocarbon radical containing 3 to 8 atoms.
• Preferred examples of cycloalkyl groups are cyclopropyl, cyclobutyl, and cyclopentyl.
• Lower alkyl groups denote C1-C6 alkyl groups and include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl, and the like.
• lower alkyl is preferably C1-C4 alkyl, and more preferably C1-C3 alkyl.
• Alkoxy denotes -O-alkyl.
• Lower alkoxy denotes -O-lower alkyl.
• “Pharmaceutically acceptable ester” refers to a conventionally esterified compound of formula I having a carboxyl group, which esters retain the biological effectiveness and properties of the compounds of formula I and are cleaved in vivo (in the organism) to the corresponding active carboxylic acid.
• esters and the use of esters for the delivery of pharmaceutical compounds is available in Design of Prodrugs. Bundgaard H ed. (Elsevier, 1985). See also, H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 108-109; Krogsgaard-Larsen, et. al., Textbook of Drug Design and Development (2d Ed. 1996) at pp. 152-191.
• “Pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases.
• Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
• Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylamrnonium hydroxide.
• Chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.
• “Pharmaceutically acceptable,” such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
• Substituted means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options.
• “Therapeutically effective amount” means an amount of at least one designated compound, that significantly inhibits proliferation and/or prevents differentiation of a human tumor cell, including human tumor cell lines.
• Compounds of the present invention as exemplified advantageously show IC50s from about 0.005 uM to about 20 uM.
• the compounds of the present invention are useful in the treatment or control of cell proliferative disorders, in particular oncological disorders. These compounds and formulations containing said compounds may be useful in the treatment or control of solid tumors, such as, for example, breast, colon, lung and prostate tumors.
• a therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
• the therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1 ,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.
• the present invention also provides pharmaceutical compositions comprising at least one compound of formula I, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier or excipient.
• Benzoic acid esters 2 are prepared using the procedures known in the art. Treatment of the cw-imidazoline 3 with phosgene in the presence of a base such as triethylamine gives the racemic carbamoyl chloride 4. Coupling of the racemic carbamoyl chloride 4 with appropriate R amine groups provides the compounds of the formula I as racemic mixtures. Many R amine groups are commercially available. If it is desired, R amine groups can be prepared using synthetic methods known in the art. Suitable processes for making these R amine groups are provided in the examples.
• the enantiomers of the carbamoyl chloride rac-4 can be separated using chiral chromatography.
• the chiral stationary phase R,R-Whelk-Ol available through Regis Technologies, can be used. Coupling of the desired enantiomer 5A with appropriate R amine groups provides the compounds of the formula I.
• optically active compounds of formula I can be obtained by chiral separation of the racemic mixtures of I.
• the chiral stationary phase Diacel ChiralPak OD or AD can be used.
• the absolute stereochemistry of the preferred enantiomer of I is determined based on the crystal structure of its complex with the human MDM2 (Vassilev et al. Science, 2004, 303, 844-848.
• the reaction mixture was diluted with ethyl acetate (1400 mL) and washed with water (2 x 600 mL), brine (1 x 200 mL), and dried over anhydrous magnesium sulfate. The solids were filtered off, and the filtrate was concentrated to dryness to give dark oil (160 g). It was taken in ethyl acetate (160 mL), and hexane (1200 mL) was added while stirring vigorously. After the mixture was settled, the supernatant was removed. The same procedure was repeated two more times with ethyl acetate and hexane. The combined supernatant was treated with charcoal then filtered. The filtrate was concentrated in vacuo.
• Methyl 4-( " cvano-dimethylmethyl)-2-ethoxy-benzoate 2-(3-Ethoxy-phenyl)-2 -methyl -propionitrile was prepared using a procedure adapted from literature (Organic Syntheses, Vol. 79, pp. 209-215).
• the reaction mixture was stirred at 100 °C for 12 hr. It was diluted with 75 mL of ethyl acetate and 75 mL of IN aqueous hydrochloric acid. The layers were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water (1 x 50 mL), brine (1 x 50 mL), and dried over anhydrous magnesium sulfate. The solids were filtered off, and the filtrate was concentrated in vacuo. The residue was dissolved in 5 mL of methylene chloride and applied to a 40 g silica gel cartridge.
• Triethylamine (3.5 mL, 25.1 mmol) was added to a solution 2-ethoxy-4-(2-hydroxy-l,l- dimethylethyl)iodobenzene (4.0 g, 12.5 mmol) in methanol (20 mL) under an atmosphere of nitrogen.
• the reaction was purged twice with carbon monoxide, and palladium (II) acetate (0.2 g, 0.9 mmol) was added.
• the reaction was stirred at 80 °C for 18 h under a positive pressure of carbon monoxide.
• the reaction was diluted with ether, washed with 10% hydrochloric acid, aqueous sodium bicarbonate, brine, dried over anhydrous magnesium sulfate and concentrated.
• Methyl 4-tert-butyl-2-(2-fluoro-ethoxy)-benzoate prepared from 4-t-butyl-2-iodophenol and 1- bromo-2-fluoro-ethane in an analogous manner as described in example 6.
• Methyl 4-tert-Butyl-2-isopropoxy-benzoate prepared from 4-t-butyl-2-iodophenol and isopropyl iodide in an analogous manner as described in example 6.
• Example 8 Methyl 4-tert-Butyl-2-isopropoxy-benzoate: prepared from 4-t-butyl-2-iodophenol and isopropyl iodide in an analogous manner as described in example 6.
• Methyl 5-(cvano-dimethylmethylV2-ethoxy-benzoate prepared from l-ethoxy-4-fluoro- benzene in an analogous manner as described in example 3.
• Methyl 5-(cvano-dimethylmethyl ' )-2-ethoxy-benzoate prepared from l-chloro-4-ethoxy-2- fluoro-benzene in an analogous manner as described in example 3.
• the progress of the reaction was monitored by thin layer chromatography (silica gel, eluting with ethyl acetate).
• the reaction mixture was then cooled in ice bath to 10 °C, Rochelle salt solution (300 mL, 1 M) was added. The ice bath was removed, and the biphasic mixture was stirred vigorously for 30 min. Ethyl acetate (300 mL) was added and stirring was continued overnight. When the layers were separated, the organic layer was decanted off. More ethyl acetate (500 mL) and Rochelle salt solution (200 mL, 1 M) were added, and the mixture were transferred to a separatory funnel. The layers were separated.
• reaction mixture was stirred at 0 °C for 30 min then concentrated to dryness.
• the orange residue was taken in methylene chloride (100 mL), and the solution was filtered through a plug of silica gel ( ⁇ 50 g). It was washed with methylene chloride (-600 mL).
• the first peak coming off the column is the desired (4S,5R)-2-(4-tgrt-butyl-2-ethoxy-phenyl)-4,5-bis-(4-chloro-phenyl)- 4,5-dihydro-imidazole- 1 -carbonyl chloride.
• Example 12 In an analogous manner as described in example 11 , there were obtained: a) C4S.5RV2-f4-tgrt-Butyl-2-ethoxy-phenyl -4.5-bis-(4-fluoro-phenvn-4.5- dihvdro-imidazole- 1 -carbonyl chloride: prepared from 2-(4-tgrt-butyl-2-ethoxy- phenyl)-4,5-bis-(4-fluoro-phenyl)-4,5-dihydro-lH-imidazole (example 10a).
• Ethanesulfonyl-piperazine prepared from 1-tert-butyloxycarbonyl-piperazine and ethylsulfonyl chloride in an analogous manner as described in example 13.
• Example 15 Ethanesulfonyl-piperazine: prepared from 1-tert-butyloxycarbonyl-piperazine and ethylsulfonyl chloride in an analogous manner as described in example 13.
• the reaction mixture was stirred for 1 h at reduced temperature.
• the solution was then made acidic with IN HCI and then diluted with 10 mL of methylene chloride.
• the vial was agitated and centrifuged.
• the organic layer was transferred to 40 L vials and concentrated in vacuo.
• the residue (1.69 g, 10.21 mmol) was diluted with lOmL of dimethylformamide.
• Piperazine-1- carboxylic acid tert-butyl ester (8.67 mmol, 0.85 eq) and diisopropylethylamine (13.27 mmol, 1.3 eq) were added.
• the reaction mixture was shaken at 65 °C overnight and concentrated in vacuo.
• N.N-Bis-(2-methoxy-ethyl)-2 -piperazin- 1 -yl-acetamide dihvdrochloride prepared from 1-tert-butyloxycarbonyl-piperazine, chloroacetylchloride and N,N-bis-(2-methoxy-ethyl)amine in an analogous manner as described in example 15.
• N-Methoxy-N-methyl-2 -piperazin- 1 -yl-acetamide dihydrochloride prepared from 1- tert-butyloxycarbonyl-piperazine, chloroacetylchloride and N-methoxy-N-methylamine in an analogous manner as described in example 15.
• N-Isopropyl-A ⁇ -methyl-2 -piperazin- 1 -yl-acetamide dihydrochloride prepared from 1- tert-butyloxycarbonyl-piperazine, chloroacetylchloride and N-isopropyl-N-methylamine in an analogous manner as described in example 15.
• Example 17 2x HCI HN N- -S0 2 e 1 -(2-Methanesulfonylethyl)piperazine dihvdrochloride Methyl vinyl sulfone (1.8 mL, 20.1 mmol) was added to a solution of ⁇ -(tert- butyloxycarbonyl)piperazine (1.50 g, 8 mmol) in methanol (84 mL). The reaction mixture was stirred at room temperature for 4 h and concentrated to a white solid.
• l-(2-Methanesulfonyl-ethyl)-[l,4]diazepane dihydrochloride was prepared from [l,4]diazepane-l -carboxylic acid tgrt-butyl ester and methyl vinyl sulfone using the same procedure as described in example 17.
• Methyl vinyl sulfone (0.42 mL, 4.8 mmol) was added dropwise to a cooled solution of N-tert- butyloxycarbonyl- 1,2-ethylenediamine hydrochloride (1.00 g, 5.1 mmol) and triethylamine (1.4 mL, 10.7 mmol) in methanol (20 mL). The reaction was stirred at room temperature for 18 h and concentrated.
• Example 22 / — ⁇ — NHS0 2 Me HN N— ' — f 2HCI N-(2-methanosulfonylethvD-piperazine dihvdrochloride Methanesulfonyl chloride (0.7 mL, 9.0 mmol) was added to a cooled solution of 4-(2-amino- ethyl)-piperazine-l -carboxylic acid tgrt-butyl ester (1.33 g, 5.8 mmol) in pyridine (25.0 mL). The reaction was stirred for 12 h and partitioned between partitioned between aqueous sodium bicarbonate and methylene chloride.
• Example 23 HCI N-( 2-Piperazin- 1 - yl-ethylV acetamide hydrochloride was prepared from 4-(2-amino-ethyl)- piperazine-1 -carboxylic acid tgrt-butyl ester and acetyl chloride in an analogous manner as describeb in example 22.
• N-r2-( , 4- ⁇ r4S.5R)-4.5-Bis-(4-chloro-phenylV2-r4-rcvano-dimethyl-methvn-2-ethoxy-phe ⁇ yn- 4.5-dihvdro-imidazole-l-carbonyl ⁇ -piperazin-l-yl)-ethyl1-methanesulfonamide was prepared from (4S,5R)-4,5-bis-(4-chloro-phenyl)-2-[4-(cyano-dimethyl-methyl)-2-ethoxy-phenyl]-4,5- dihydro-imidazole-1 -carbonyl chloride (example 12j) and N-(2-methanosulfonylethyl)- piperazine hydrochloride (example 22) in an analogous manner as described in example 25.
• 4,5-bis-(4-chloro-phenyl)-2-[2-ethoxy-4-(2 -methoxy- 1 , 1 -dimethyl-ethyl)-phenyl]-4,5-dihydro- lH-imidazole (580 mg, 1.166 mmol) was reacted with phosgene (725 uL, 1.399 mmol, 20% solution in toluene) using the procedure as described in example 12 to give 4,5-bis-(4-chloro- phenyl)-2-[2-ethoxy-4-(2 -methoxy- 1 , 1 -dimethyl-ethyl)-phenyl]-4,5-dihydro-imidazole- 1 - carbonyl chloride (370 mg, 57% yield) as white solids.
• 4,5-Bis-(4-chloro-phenyl)-2-[2-ethoxy-4-(2-methoxy-l,l-dimethyl-ethyl)-phenyl]-4,5-dihydro- imidazole-1 -carbonyl chloride 60 mg, 0.107 mmol
• 2-piperazinone 16.1 mg, 0.161 mmol
• 4,5-bis-(4-chloro-phenyl)- 2-[2-ethoxy-4-(2-methoxy- 1 , 1 -dimethyl-ethyl)-phenyl]-4,5-dihydro-imidazole- 1 -carbonyl ⁇ - piperazin-2-one (64.7 mg) as white solids.
• 4,5-Bis-(4-chloro-phenyl)-2-[2-ethoxy-4-(2 -methoxy- 1 , 1 -dimethyl-ethyl)-phenyl]-4,5-dihydro- imidazole-1 -carbonyl chloride 60 mg, 0.107 mmol
• l-mo ⁇ holin-4-yl-2- piperazin- 1-yl-ethanone 34.2 mg, 0.161 mmol, Oakwood Products) using the procedure described in example 25 to give 4,5-bis-(4-chloro-phenyl)-2-[2-ethoxy-4-(2 -methoxy- 1,1- dimethyl-ethyl)-phenyl]-4,5-dihydro-imidazole- 1 -carbonyl ⁇ -piperazin- 1 -yl)- 1 -mo ⁇ holin-4-yl- ethanone (70.1 mg) as white solids.
• N,/V-diethyl-2-(4-iodo-3-methoxy- phenyl)-isobutyramide (361 mg, 0.927 mmol) was converted to 4-(l-diethylcarbamoyl-l- methyl-ethyl)-2-methoxy-benzoic acid methyl ester (166 mg, 56% yield) as yellow oil.
• the ability of the compounds to inhibit the interaction between p53 and MDM2 proteins was measured by an HTRF (homogeneous time-resolved fluorescence) assay in which recombinant GST-tagged MDM2 binds to a peptide that resembles the MDM2-interacting region of p53 (Lane et al.). Binding of GST-MDM2 protein and p53-peptide (biotinylated on its N-terminal end) is registered by the FRET (fluorescence resonance energy transfer) between Europium (Eu)-labeled anti-GST antibody and streptavidin-conjugated Allophycocyanin (APC).
• FRET fluorescence resonance energy transfer
• Test is performed in black flat-bottom 384-well plates (Costar) in a total volume of 40 uL containing:90 nM biotinylate peptide, 160 ng ml GST-MDM2, 20 nM streptavidin-APC (PerkinElmerWallac), 2 nM Eu-labeled anti-GST-antibody (PerkinElmerWallac), 0.2% bovine serum albumin (BSA), 1 mM dithiothreitol (DTT) and 20 mM Tris-borate saline (TBS) buffer as follows: Add 10 uL of GST-MDM2 (640 ng/ml working solution) in reaction buffer to each well.
• IC 50 S showing biological activity that applies to compounds of the subject matter of this invention ranges from about 0.005 uM to about 2 uM. Specific data for some examples are as follows:

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